Activated polyethylene glycol compounds

ABSTRACT

Novel arylisothiocyanate compounds are described that are useful for activating alcohol-containing macromolecules, for example polyethyleneglycols and cellulose, for covalent linkage to amino-groups of biomolecules, for example polypeptides such as antibodies, enzymes, and proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of allowed U.S. patent applicationSer. No. 09/105,644 filed Mar. 25, 2002.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to a novel compound and method forpreparation of polyethylene glycol (PEG) adducts of biomolecules, andparticularly to PEG adducts of proteins and peptides.

[0003] Attachment of large macromolecules such as polyethylene glycol(PEG) to biomolecules such as proteins or peptides via chemicalattachment is desired for modification of the properties of the proteinsor peptides. Linking to PEG is referred to in the art as “pegylation”.Biomolecules circulating in the blood outside of a cell are subject toclearance, and can move through blood vessel walls(extravascularization). Attachment of relatively small biomolecules tolarge macromolecules can reduce extravascularization, and can enhancethe in-vivo circulation half-life of the biomolecule.

[0004] Increasing half-life of the biomolecule in circulation isparticularly important when the biomolecule is intended for therapeuticuse. Pegylation of certain biomolecules reduces kidney clearance andspurious enzymatic degradation and immune system recognition. In“Artificial Blood”, Science, 295:1002, 1004-1005 (Feb. 8, 2002), JerryE. Squires cites literature reports that conjugation of hemoglobin tomacromolecules such as dextran, polyethylene glycol or polyoxyethyleneretards the rate at which cell-free hemoglobin is cleared from bloodcirculation, extending intravascular dwell time up to 48 hours. Thealteration of the effective solution volume of the hemoglobin throughlinkage to a macromolecule alters the colligative properties ofcell-free hemoglobin, including osmotic pressure that appears to have asignificant effect on blood pressure.

[0005] Polyethylene glycol is approved by the U.S. Food and DrugAdministration for internal and topical use due to its low toxicity.Additional utilities and features of PEG-biomolecule conjugates aredescribed in Polyethylene Glycol Chemistry: Biotechnical and BiomedicalApplications, J. M. Harris, ed. Plenum, N.Y., 1992 and PolyethyleneGlycol Chemistry and Biological Applications, J. M. Harris and S.Zalipsky, eds., ACS, Washington, 1997.

[0006] A requisite for preparation of polyethylene glycol conjugates ofbiomolecules is a suitably activated PEG molecule that under properconditions reacts with a target biomolecule in an efficient, predictablemanner such that the native activity of the target biomolecule is notadversely affected to a significant degree. For sensitive targetbiomolecules such as proteins and peptides, reactions to form PEGconjugates are best conducted in aqueous, buffered systems in order toavoid denaturation and concomitant loss of biological activity.

[0007] Most, if not all, activated polyethylene glycol compoundsdescribed in the patent and scientific literature and intended forconjugation to biomolecules such as proteins react with water inaddition to the target biomolecule. See, for example, Scheme I below.Typically, the conundrum of derivatizing a target biomolecule in anaqueous medium with a water-sensitive, activated, polyethylene glycolreagent is partially solved by employing a large excess (5-10 fold) ofreagent, while maintaining rigorous control of pH and temperature. Anobjective of any pegylation procedure is to produce pegylatedbiomolecules with stable, enhanced physiological properties in apredictable and reproducible manner on a meaningful, economical scale.

[0008] Scheme 1, below represents a reaction between an activatedpegylation reagent of the art and a biomolecule reactant having acidichydrogens (hydrido groups). The scheme shows the formation of hydrolysisproduct, pegylated biomolecule and protonated leaving group (HA). Asillustrated in Scheme 1, activated pegylation reagents described in thecurrent art often involve production of a leaving group (HA in Scheme 1below) in addition to any hydrolyzed PEG reagent (shown as PEG-OH). Theproduction of a leaving group presents an additional workup andpurification problem during the isolation of the PEG-modified targetmolecule. The extent of the isolation/purification problem is influencedby the magnitude of the excess reagent employed in order to achieve thedesired level of PEG modification which, in turn, is a function ofhydrolysis rate of the reagent in water and the coupling rate onto aresidue of the target molecule (typically —NH₂ or —SH).

[0009] Typically, activated polyethylene glycol reagents described andused in the current art are acylating reagents directed toward primaryamine residues (—NH₂) in the target biomolecule. A common type of PEGacylating reagent is the so-called “active ester” derivative of PEG.Active ester PEGs of the N-hydroxysuccinimide (NHS), hydroxybenztriazole(HBT), imidazole (IM)and p-nitrophenol (PNP) have been described and arecommercially available. (See Shearwater Corporation, Catalog 2001,Huntsville, Ala. 35801, www.shearwatercorp.com).

[0010] Reagents of the type

[0011] mPEG-R—C(O)—OX,

[0012] where R=(O)C—(CH₂)_(y) or (CH₂)_(y) or (—O—),

[0013] y=zero through 4, and

[0014] X=NHS, HBT, IM or PNP

[0015] exhibit half-lives of hydrolysis of 1 minute (or less) toapproximately 24 minutes at pH=8 and 25° C. Further, as half-life goesup, reactivity goes down. Recommended excesses of PEG acylating reagentvary from equal mass to 10-fold mass relative to target molecule(Shearwater Catalog 2001 p.12). Depending on the molecular weight of thetarget biomolecule, this recommended mass excess can be greater than a100-1000 molar excess.

[0016] Incorporation of macromolecules such as PEG into biomolecules byusing currently-available PEG acylating reagents is a demonstrablyinefficient process. Problems associated with the acylating PEG reagentsof the current art are exacerbated on a large scale. Variables affectingefficiency and reproducibility of pegylation procedures based on currentart acylating reagents include: half-life (t_(1/2)) of hydrolysis, pHvalue, temperature, time, mixing rate, nature and toxicity of leavinggroup, ease of product purification from leaving groups and hydrolyzedreagent, as well as the rate and extent of reactivity of the reagenttoward the target biomolecule.

[0017] The rapid hydrolysis rates of acylating PEG reagents employed bythe standard art preclude practical application of multi-functional,crosslinking pegylation reagents of the following type, where A is aleaving group.

[0018] A multifunctional, activated PEG reagent as shown above is usefulfor establishing intramolecular cross-links within protein molecules forthe purpose of mapping sub-unit geographies and for stabilization ofprotein configurations and activities. Rapid hydrolysis rates ofstandard art PEG reagents, used at large molar excess favor apreponderance of “one on hits”, where one carboxyl end of thebifunctional molecule links to the target protein but the other endhydrolyzes to a carboxylic acid instead of also linking to the protein,and forming stabilizing cross-links.

[0019] In a significant advance, workers at the Albert Einstein Collegeof Medicine of Yeshiva University N.Y., University of Iowa andBioAffinity Systems describe a novel approach for activating PEG forattachment to biomolecules that largely circumvents problems associatedwith hydrolytically unstable reagents. As disclosed in Acharya et al.U.S. Pat. No. 5,585,484, U.S. Pat. No. 5,750,725, U.S. Pat. No.6,017,943, entitled “Hemoglobin Crosslinkers”, and Belur N. Manjula, etal., J. Biol. Chem., 275(8):5527-5534 (2000), a maleimide-activated PEGreagent is employed to form stable thioether bonds with an indigenous oradded sulfhydryl moiety in the biomolecule. The maleimide functionreacts rapidly with —SH groups without significant hydrolysis at pH6.5-7.0 and without the production of a leaving group as is shown in thereaction below, wherein R—SH is a sulfhydryl-containing biomolecule.

[0020] Biomolecules such as a protein having a paucity of —SH groupsmust first be reacted with a thiolating reagent such as 2-iminothiolane(or the like) to convert native —NH₂ groups from lysine into —SH groups.A practical drawback with the maleimide reagents is that they aredifficult molecules to obtain synthetically.

[0021] The chemical formula of phenyl isothiocyanate, also known asisothiocyanatobenzene or isothiocyanic acid phenyl ester is shown below.

[0022] As noted in the Merck Index, 11^(th) Ed., Susan Budavari, et al.,eds., Merck & Co., Inc. (Rahway, N.J.: 1989), p. 7275, phenylisothiocyanate is known to be used as a derivatizing agent for primaryand secondary amines. Such derivatization of primary and secondaryamines has typically been used for carrying out Edman degradation andamino acid analyses by HPLC.

[0023] This art does not teach the introduction of macromolecules suchas polyethylene glycol by the use of a phenylisothiocyanate-containingmolecule. Nor are there available any commercial sources of polyethyleneglycols or other such macromolecules activated withphenylisothiocyanate. Furthermore, the precursors for obtaining suchactivated macromolecules are not commercially available or known in theart, e.g. agents containing both a isothiocyanate group and anisocyanate group.

[0024] The direct linkage of an alcohol-containing polysaccharide to anamine-containing protein vie reductive amination is known for linkingantigenic polysaccharides to carrier proteins for the preparation ofvaccines. Aldehyde groups are prepared on either the reducing end [Porenet al. (1985) Mol. Immunol., 22:907-919] or the terminal end [Andersonet al. (1986) J. Immunol., 137:1181-1186] of an alcohol-containingoligosaccharide or relatively small polysaccharide, which are thenlinked to an amine group in the protein via reductive amination. U.S.Pat. No. 4,356,170 discloses such preparation of useful polysaccharidesthat are reduced and then oxidized to form compounds having terminalaldehyde groups that can be reductively aminated onto free amine groupsof carrier proteins such as tetanus toxoid and diphtheria toxoid with orwithout significant cross-linking. Several of the problems associatedwith the attachment of biomolecules to macromolecules are overcome byuse of the reagents and processes described hereinafter.

BRIEF SUMMARY OF THE INVENTION

[0025] The present invention provides reagents and processes for thelinking of alcohol-containing macromolecules, M, to amine-containingbiomolecules, B. The compositions of the present invention are linkingreagents, linking reagent precursors and reacted linking reagents having(i) an isothiocyanate or a thiourea derivative of an amine-containingbiomolecule B, and (ii) one or two other phenyl substituents in themeta- or para-position that is (a) isocyanate, (b) acylazide, or (c) aurethane derivative of an alcohol-containing macromolecule M. Examplesof alcohol-containing macromolecules, M, include but are not limited topolysaccharides and hydroxylated silica derivatives. Examples ofamine-containing biomolecules, B, include but are not limited to nucleicacids and polypeptides.

[0026] A general chemical formula for a linking reagent or linkingreagent precursor is shown below having (i) isothiocyanate, and (ii) oneor two other phenyl substituents in the meta- or para-position that is(a) isocyanate, (b) acylazide, or (c) a urethane derivative of analcohol-containing macromolecule M.

[0027] The subscript, n, is 1 or 2, denotes the number of R substituentson the phenyl ring. R is —NHC(O)—O—M, —NCO or —C(O)N₃., with R of —NCOor —C(O)N₃ in linking reagent precursors. M is a reactedalcohol-containing macromolecule. The R phenyl substituents are in themeta-, di-meta- or para-positions relative to the isothiocyanate group.The di-meta di-substituted phenylisothiocyanate is preferred over themeta, para-di-substituted phenylisothiocyanate.

[0028] A general formula for a reacted linking reagent of the presentinvention is shown below having (i) a thiourea derivative that is areaction product of an amine-containing biomolecule, B, and anisothiocyanate, and (ii) one or two other phenyl substituents in themeta- or para-position that is a urethane derivative of analcohol-containing macromolecule, M. The subscript n is 1 or 2, asbefore.

[0029] The isothiocyanate moiety of the linking reagent of the inventionreacts with a primary amino group of a biomolecule B to be linked usingthe linking reagent, to form a thiourea moiety. In the balanced chemicalreaction, there is effectively no leaving group from the reaction of theisothiocyanato group with the amino group. Thus, in some embodiments,purification issues due to reaction side products and the costsassociated therewith are eliminated.

[0030] Some embodiments of linking reagent precursors are chemicallyfacile and relatively inexpensive to prepare. Some embodiments of thelinking reagent precursors are stable enough for preparation andshipment with a reasonable shelf life.

[0031] In some embodiments of the invention, linking reagents are usefulfor increasing the hydrodynamic volume of biomolecules, which mayprolong the half-life of a biomolecule circulating in blood in a livingbody. In some embodiments, linking reagents are useful for linking abiomolecule to a surface.

[0032] The various embodiments of the present invention has severalbenefits and advantages, however all embodiments do not necessarilyprovide all of the below-listed benefits and advantages. Furtherbenefits and advantages of embodiments of the invention will berecognized by workers in the art.

[0033] One benefit of several embodiments of the invention is that itprovides an embodiment of a novel, activated pegylation reagent thatpermits rapid, efficient, one-step production of pegylated biomoleculeshaving stable, enhanced physiological properties in a predictable andreproducible manner on a meaningful, economical scale.

[0034] An advantage of several embodiments of the invention is that itprovides an embodiment of a reagent that does not suffer from asignificant hydrolysis rate in aqueous reaction media that are bufferedat a pH value of about 5.5 to about 8.5, maintains good rates ofreactivity, is specific for primary amine groups in target biomoleculesand does not produce a leaving group upon covalent attachment to thetarget biomolecule.

[0035] A further benefit of several embodiments of the invention is thatit provides an embodiment that permits direct PEG modification of one ormore amino (preferably —NH₂) groups or moieties on the biomoleculewithout first converting those amino groups to one or more sulfhydryl(—SH) groups.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention provides reagents and processes for thelinking of alcohol-containing macromolecules, M, to amine-containingbiomolecules, B. The compositions of the present invention are linkingreagents, linking reagent precursors and reacted linking reagents having(i) an isothiocyanate or a thiourea derivative of an amine-containingbiomolecule B, and (ii) one or two other phenyl substituents in themeta- or para-position that is (a) isocyanate, (b) acylazide, or (c) aurethane derivative of an alcohol-containing macromolecule M. Examplesof alcohol-containing macromolecules, M, include but are not limited topolysaccharides and hydroxylated silica derivatives. Examples ofamine-containing biomolecules, B, include but are not limited to nucleicacids and polypeptides.

[0037] A general chemical formula for a linking reagent or linkingreagent precursor is shown below having (i) isothiocyanate, and (ii) oneor two other phenyl substituents in the meta- or para-position that is(a) isocyanate, (b) acylazide, or (c) a urethane derivative of analcohol-containing macromolecule M.

[0038] The subscript, n, is 1 or 2, denoting the number of Rsubstituents on the phenyl ring. R is —NHC(O)—O—M, —NCO or —C(O)N₃. M isa reacted alcohol-containing macromolecule. The R phenyl substituentsare in the meta-, di-meta- or para-positions relative to theisothiocyanate group.

[0039] A general chemical formula for a linking reagent precursor havingisothiocyanate and isocyanate moieties is shown below, with n being 1 or2, as before.

[0040] A general chemical formula for a linking reagent precursor havingisothiocyanate and the precursor to the isocyanate, an acyl azide, isshown below, with n being 1 or 2, as before.

[0041] A general formula for a linking reagent that is a kind ofactivated alcohol-containing macromolecule is shown below, having anisothiocyanate group and —NHC(O)—O—M, where M is a reactedalcohol-containing macromolecule, with n being 1 or 2, as before. In apreferred embodiment where n is 1, the isothiocyanate moiety is in the4-position (para) relative to the —NHC(O)—O—M.

[0042] The isothiocyanate moiety of the linking reagent of the inventionreacts with a primary amino group (H₂N—B) to form a thiourea moiety, asshown in Scheme 2 below. In the balanced chemical reaction, there iseffectively no leaving group from the reaction of the thiocyanato groupwith the amino group. The linking reagent can also react with asecondary amine.

[0043] Primary amines present in a typical protein are part of a lysine(Lys or K; amino pK=10.5) amino acid residue, and also the aminoterminus of the peptide backbone.

[0044] A general formula for a reacted linking reagent of the presentinvention is shown below having (i) a thiourea derivative that is areaction product of an amine-containing biomolecule, B, and anisothiocyanate, and (ii) one or two other phenyl substituents in themeta- or para-position that is a urethane derivative of analcohol-containing macromolecule, M. The subscript n is 1 or 2, asbefore.

[0045] Also contemplated is an alcohol-containing macromolecule, M, thatis derivatized with more than one phenylisothiocyanate group. A generalformula shown below illustrates a macromolecule having twophenylisothiocyanate groups. Such a molecule is useful for cross-linkingtwo biomolecules, B, or two amino groups within a biomolecule.

[0046] A preferred form of the reagent for a non-crosslinking pegylationreagent, represented by the formula shown below, is a phenylisothiocyanate derivative of methoxy-polyethylene glycol (mPEG).

[0047] The methoxy-PEG moiety in the formula above is represented by—(O)—CH₂CH₂—(OCH₂CH₂)_(x)—O—CH₃, where x is an average repeat unitnumber that is about 5 and about 500, preferably about 50 to about 300.

[0048] A preferred form of the reagent for a crosslinking pegylationreagent, represented by the formula shown below, is adi-(phenylisothiocyanate) derivative of PEG.

[0049] The PEG moiety in the formula above is represented by—(O)—CH₂CH₂—(OCH₂CH₂)_(x)—O—, where x is an average repeat unit numberthat is about 5 and about 500, preferably about 50 to about 300.

[0050] A preferred form of the reagent for a linking pegylation reagentwhere n is 2, represented by the formula shown below, is a di-metapegylated derivative of phenylisothiocyanate.

[0051] The mPEG moiety in the formula above is represented by—(O)—CH₂CH₂—(OCH₂CH₂)_(x)—O—CH₃, where x is an average repeat unitnumber that is about 5 and about 500, preferably about 50 to about 300.

[0052] The isothiocyanate moiety of the linking reagent of the inventionreacts with a primary amino group of a biomolecule B to be linked usingthe linking reagent, to form a thiourea moiety. In the balanced chemicalreaction, there is effectively no leaving group from the reaction of theisothiocyanato group with the amino group. The linking reagent can alsoreact with a secondary amine. Preferably, for a protein, the primaryamine is from the side chain of lysine or the amino terminus.

[0053] A contemplated biomolecule may be a polypeptide such as anantibody, enzyme, or protein, or a nucleic acid. Some exemplarypolypeptides that benefit from pegylation include, but are not limitedto, hemoglobin, bilirubin oxidase and insulin. Several contemplatedbiomolecules are discussed in Polyethylene Glycol Chemistry:Biotechnical and Biomedical Applications, J. M. Harris, ed. Plenum,N.Y., 1992 and Polyethylene Glycol Chemistry and BiologicalApplications, J. M. Harris and S. zalipsky, eds., ACS, Washington, 1997.In another embodiment, the invention contemplates the linkage of theisothiocyanate to an amine of a biomolecule that is not a typicalpeptide residue. The invention contemplates the linking of anamine-containing biomolecule such as a drug or pro-drug, a hapten, acytokine, a ligand for a receptor, a peptide analog, a nucleic acid baseor nucleic acid analog.

[0054] In a method for providing a pegylated protein, anamine-containing protein is linked to a large, alcohol-containing PEGmacromolecule, such as a PEG-phenyl isothiocyanate compound. Such apegylated protein provides a protein that can circulate in the bloodwith a longer half-life than the non-pegylated protein.

[0055] In a method for providing an antibody linked to a surface, anamine-containing antibody is linked to an alcohol-containing cellulosederivatized with a phenyl isothiocyanate compound. Such a linkedantibody is useful, for example in methods where separation betweenmaterial that binds to the antibody from material that does not bind tothe antibody is desired.

[0056] In a method for providing a protein linked to a surface, anamine-containing antibody is linked to an alcohol-containing cellulosederivatized with a phenyl isothiocyanate compound.

[0057] In a method for providing a ligand linked to a surface, anamine-containing ligand is linked to an alcohol-containing surface thathas been derivatized with a phenyl isothiocyanate. Such a linked ligandis useful, for example in methods where separation between material thatbinds to the ligand from material that does not bind to the ligand isdesired. Such an alcohol-containing surface might be a cellulosemembrane or a silica bead having reactive hydroxyl groups.

[0058] In a method for providing a multi-subunit protein with enhancedstability, amine-containing proteins are crosslinked intramolecularlywith a bifunctional alcohol-containing molecule, such as a PEGdi-(phenylisothiocyanate) compound. Such a cross-linked multisubunitprotein is useful, for example in studies of the relationships betweensubunits or to ascertain what proteins are in a complex, such as atranscription complex with effectors.

[0059] In an embodiment where n is 1, the R group is preferably in thepara position. Thus, in a preferred embodiment where n is 1, theisothiocyanate moiety is in the 4 position (para) relative to theisocyanate (—NCO), —C(O)N₃, or —NHC(O)—O—M moiety.

[0060] In an embodiment where n is 2, there are two R substituents onthe phenyl ring. The di-meta di-substituted phenylisothiocyanate ispreferred over the meta, para-di-substituted phenylisothiocyanate. In anembodiment where n is 2, the invention contemplates R groups that arenot identical, such as different M groups in —NHC(O)—O—M, or an —NCOgroup and an —NHC(O)—O—M group. Thus, reaction of less than 100 percentof the —NCO to form —NHC(O)—O—M is contemplated, as is the use of amixture of macromolecular forms (e.g. PEG that has a range of chainlengths, as is typical in some commercially available PEG preparations).

[0061] The azido compound, where R is —C(O)N₃, is stable, and iscontemplated for use as a general precursor linking reagent. When R is—NHC(O)—O—M, M is an alcohol-containing macromolecule is derivatizedwith one or more, but preferably only one or two, phenylisothiocyanategroups. Where M is derivatized with more than one phenylisothiocyanategroup, the reagent is a crosslinking reagent. Where M is PEG, and M isderivatized with more than one phenylisothiocyanate group, the reagentis a crosslinking pegylation reagent.

[0062] The invention contemplates the linkage of the isothiocyanategroup to an amine, preferably a primary amino group of a biomolecule Bto be linked using the linking reagent. Such a biomolecule is preferablya nucleic acid or a polypeptide, e.g. an antibody, enzyme, or protein.

[0063] In an embodiment, the invention contemplates the linkage of theisothiocyanate to an amine of a biomolecule that is not a typicalpeptide residue. The invention contemplates the linking of anamine-containing biomolecule such as a drug or pro-drug, a cytokine, aligand for a receptor (e.g. example streptavidin), a peptide analog, anucleic acid base or nucleic acid analog.

[0064] In a method for providing a pegylated polypeptide, anamine-containing polypeptide is linked to a large, alcohol-containingPEG macromolecule, such as a PEG-phenyl isothiocyanate compound. Such apegylated polypeptide provides a polypeptide that can circulate in theblood with a longer half-life than the non-pegylated form. Such apegylated polypeptide is thus useful in a method of treating a mammal(including homo sapiens) involving the administration of a polypeptide.

[0065] The invention contemplates a method of making a stabilizedpeptide through attachment of a polyethylene glycol to a peptide. In apreferred embodiment, a bifunctional molecule (a phenyl group with anisothiocyanate moiety and an isocyanate moiety) serves to crosslink thepolyethylene glycol moiety to a peptide via an amine group, preferably aprimary amino group, such as on a lysine side chain.

[0066] A polyethylene glycol (PEG) compound can itself be quite variedin composition, but contains at least one poly(oxyethylene) chain[(—CH₂CH₂O—)_(x)]having an average molecular weight of about 300 (x is5) to about 22,000 (x is 500), with an average molecular weight of about2,300 (x is 50) to about 13,300 (x is 300) being more preferred. Morespecifically, the reacted PEG compound group M of the linker correspondsto the formula —CH₂CH₂—(CH₂CH₂O)_(n)—CH₂CH₂Y where X, n and R aredefined and discussed hereinbelow.

[0067] In the above formula, x is a number having an average value ofabout 5 to about 500, and more preferably about 50 to about 300. It iswell known that the higher molecular weight PEG compounds are usuallymixtures rather than pure compounds having a single molecular weight. Asa result, x, the number of ethyleneoxy repeating units, is a number thatis an average number. The terminal Y group is —OH or a C₁-C₁₀hydrocarbyl ether (alkoxy group) having a molecular weight of up toabout one-tenth of the —(CH₂CH₂O)_(n)— portion.

[0068] Exemplary C₁-C₁₀ hydrocarbyl ether groups are well known andinclude alkyl, alkenyl, alkynyl and aromatic ethers. Illustrative C₁-C₁₀ethers thus include methyl, which is most preferred, ethyl, isopropyl,n-butyl, cyclopentyl, octyl, decyl, 2-cyclohexenyl, 3-propenyl, phenyl,1-naphthyl, 2-naphthyl, benzyl, phenethyl and the like ethers. Theseether groups can also be named methoxy, ethoxy, isopropoxy, n-butoxy,cyclopentyloxy, octyloxy, decyloxy, 2-cyclohexenyloxy, 3-propenyloxy,phenoxy, 1-naphthoxy, 2-naphthoxy, enzyloxy and phenethyloxy. A C₁-C₆hydrocarbyl group is a particularly preferred Y group.

[0069] The molecular weight of a C₁-C₁₀ hydrocarbyl ether can be up toabout one-tenth of the weight of the —(CH₂CH₂O)_(x)— portion of the PEGgroup. Thus, where x is 20, the —(CH₂CH₂O)_(n)— portion has a molecularweight of 880 (20×44) so that the molecular weight of Y can be up toabout 90, or about the weight of a phenoxy group. It is more preferredthat the molecular weight of the C₁-C₁₀ hydrocarbyl group be about 0.2to about 2 percent of the molecular weight of the —(CH₂CH₂O)_(x)—portion.

[0070] The linker molecules of the invention are useful in a variety ofmethods and assays involving amine-containing peptides or proteins. Theinvention can be used so that detection enzymes are the amine-containingbiomolecule B that is linked to a macromolecule or surface, M, such ascellulose, for use in an assay. Skilled workers in the art canappreciate other methods of using the linker of the invention in theirassays with their own amine-containing biomolecules, B, andalchohol-containing macromolecules, M.

[0071] For example, streptavidin, such as the wild type or mutantstaught in U.S. Pat. No. 6,312,916 granted Nov. 6, 2001, can be useful inbinding biotinylated molecules. The streptavidin is linked to amacromolecule, such as PEG, which can change a molecular weight cut offand permit dialysis-type binding assays. The streptavidin B is linked toa macromolecule such as a cellulose membrane, M, which can then bewashed with solutions that may contain biotinylated molecules.

[0072] Contemplated hydroxy-containing surfaces include, but are notlimited to, appropriately derivatized silica, cellulose or gold.

[0073] Contemplated hydroxy-containing macromolecules includepolysaccharides. On large polysaccharides, one or more of the hydroxygroups may be reacted with a linking reagent precursor. Reaction with adi-meta compound may result in crosslinking of a polysaccharide chain.The carbohydrate itself can be synthesized by methods known in the art,for example by enzymatic glycoprotein synthesis as described by Witte etal. (1997) J. Am. Chem. Soc., 119:2114-2118.

[0074] Several oligosaccharides, synthetic and semi-synthetic, andnatural, are discussed in the following paragraphs as examples ofoligosaccharides that are contemplated haptens to be used in making aHBc conjugate of the present invention. U.S. Pat. No. 4,220,717 alsodiscloses a polyribosyl ribitol phosphate (PRP) hapten for Haemophilusinfluenzae type b. Andersson et al., EP-0 126 043-Al, disclosesaccharides that can be used in the treatment, prophylaxis or diagnosisof bacterial infections caused by Streptococci pneumoniae. EuropeanPatent No. 0 157 899-B1, the disclosures of which are incorporatedherein by reference, discloses the isolation of antigenic pneumococcalpolysaccharides.

[0075] The optimal ratio of macromolecule polysaccharide M tobiomolecule B in the linked form depend on the particularpolysaccharide, the biomolecule, and the linker molecule used.

[0076] In a method for providing an antibody linked to a surface, anamine-containing antibody is linked to an alcohol-containing cellulosederivatized with a phenyl isothiocyanate compound or otherhydroxy-containing surface. Such a linked antibody is useful, forexample in methods where separation between material that binds to theantibody from material that does not bind to the antibody is desired.

[0077] In a method for providing a protein linked to a surface, anamine-containing antibody is linked to an alcohol-containing cellulosederivatized with a phenyl isothiocyanate compound.

[0078] In a method for providing a ligand linked to a surface, anamine-containing ligand is linked to an alcohol-containing cellulosederivatized with a phenyl isothiocyanate. Such a linked ligand isuseful, for example in methods where separation between material thatbinds to the ligand from material that does not bind to the ligand isdesired.

[0079] In a method for providing a multi-subunit protein with enhancedstability, amine-containing proteins are crosslinked intramolecularlywith a bifunctional alcohol-containing molecule, such as a PEGdi-(phenylisothiocyanate) compound. Such a cross-linked multisubunitprotein is useful, for example in studies of the relationships betweensubunits or to ascertain what proteins are in a complex, such as atranscription complex with effectors. Also contemplated is linkingbetween different molecules in an associated complex, for exampletranscription factors with RNA polymerase.

[0080] A preferred pegylation reagent according to the invention is madeusing methods known in the art or their equivalents.

[0081] In one example of the invention, the preparation of a PEGmolecule having a phenyl isothiocyanate activating group is carried outas shown in Scheme 3, below. Briefly, a para-aminobenzoic acid isreacted with thiophosgene to produce 4-carboxyphenyl isothiocyanate, asdescribed in Example 1. The carboxy moiety is activated as the azide toform isothiocyano benzoyl azide by known methods, such as that describedin Example 2. The azide-activated carboxy moiety was then heated tocause internal rearrangement to isocyanophenyl isothiocyanate, by theCurtius Rearrangement described in Example 3.

[0082] The invention contemplates linking reagents having one equivalentof hydroxy-containing macromolecule. A contemplated linking reagent isnot limited to a para-substituted phenyl isothiocyanate. Meta- anddi-meta-substituted phenyl isothiocyanate compounds are alsocontemplated. The synthesis of the para-substituted compound is shownbelow in Examples 1-3, starting with para-aminobenzoic acid. Acorresponding meta-substituted aminobenzoic acid compounds arecommercially available.

[0083] The invention contemplates bifunctional linking reagents havingmore than one phenyl isothiocyanate groups, which are useful ascrosslinking reagents. Using the azide-activated synthetic isocyanatereaction described above, a hydroxy-containing macromolecule is linkedto the phenyl isothiocyanate group. The use of a macromolecule with morethan one hydroxy group, and appropriate adjustments of stoichiometry andreaction conditions, results in a reagent that has more than one phenylisothiocyanate group. For example, a bifunctional PEG reagent is madeusing a PEG diol, as illustrated by Example 4, below. Such bifunctionallinking reagents are useful for linking two amine-containingbiomolecules (the same or different biomolecules).

[0084] The invention contemplates linking reagents having one or morehydroxy-containing macromolecules on a single phenyl isothiocyanategroup. Such reagents are made using the analogous procedures to thosedescribed herein in detail for the mono-substituted phenylisothiocyanate compound. For example, a di-meta reagent is made startingwith 5-aminoisophthalic acid, commercially available (e.g. AldrichProduct No. 18,627-9). The conversion of the amino group toisothiocyanate then proceeds as described in the Examples below, usingCSCl₂, NaOAc and H₂O, using methods known in the art.

[0085] A preferred method of making a di-meta reagent is shown in Scheme4 below. Commercially available amino isophthalic acid serves as thestarting material that is converted to the corresponding isothiocyanatecompound, 3,5-dicarboxyphenyl isothiocyanate, using thiophosgene,C(S)Cl₂, in the presence of an aqueous solution of sodium acetate asillustrated below in Example 5. The two carboxylic acid moieties arethen activated with sodium azide in the presence of phenyldichlorophosphate and pyridine, as illustrated in Example 6 below, toprovide the corresponding acylazido phenylisothiocyanate. The acylazidomoieties convert smoothly to isocyanate moieties via the Curtiusrearrangement, which then react with an alcohol-containingmacromolecule, such as PEG, to provide a contemplateddi-meta-substituted phenylisothiocyanato reagent.

[0086] The linking of two hydroxy-containing macromolecules to thedi-meta-substituted reagent also proceeds by methods known in art,examples of which are provided hereinbelow. Preferably, one hydroxygroup from each of two macromolecules reacts with a single di-metaisocyanate-substituted phenyl isothiocyanate compound to form a di-metalinking reagent.

[0087] Thus is it recognized that the methods described herein areuseful to activate a variety of hydroxy-containing macromolecules,including hydroxy and polyhydroxy compounds. Contemplated examplesinclude but not limited to methoxy PEG, PEG-diols and branched PEGs ofvarious molecular weights. Hydroxy and polyhydroxy compounds other thatpolyethylene glycols are contemplated, including but not limited tocelluloses and starches. Such reagents adapting methods known in the artfor reacting phenylisocyanates with hydroxl-containing molecules, suchas described herein adapted for the molecules of interest, by adjustingthe amount of p-isothiocyanobenzoylazide added to match the correctstoichiometry of macromolecular hydroxyls present. Normally, astoichiometric or slight excess (zero to ten percent molar excessrelative to the hydroxy; where zero percent excess is a one-to-one molarratio) of the azide is added. For instance, dry, insoluble surfaces,i.e., cellulose bearing a plurality of primary hydroxyl groups isactivated by soaking the surface in a solution of p-isothiocyanophenylisocyanate at 20-60° C. (prepared in situ), as described in Example 5,below, or hydroxy-derivatized silica. The activated surface is ready forlinking to an amine-containing molecule, useful for a wide variety ofapplications.

[0088] The activated supports thus obtained are useful for immobilizingfunctional proteins such as enzymes or antibodies under mild conditions(e.g. pH 7.4-8.25 10 mm bicarbonate buffer). Likewise, immunoconjugatesof small, hydroxy-containing haptens, e.g. vitamin B-12,hydroxyprogesterone, and digoxigenin, are made utilizing a contemplatedisothiocyanophenyl isocyanate. A protocol for linking small,hydroxy-containing molecules to an isocyanate compound is described inM. E. Annunziato, et al., Bioconjugate Chem., 4:212-218 (1993), thedisclosures of which are incorporated in full herein by reference.

[0089] The phenyl isothiocyanate moiety is stable against hydrolysis inaqueous buffers, and it maintains excellent rates of reactionspecifically with primary amines in target biomolecules. Bi- andmulti-functional linking reagents are thus possible and practical forthe efficient derivatization of target molecules for the purpose ofestablishing inter and/or intra molecular crosslinks which stabilizenative tertiary structure.

EXAMPLE 1 Synthesis of 4-Carboxyphenyl Isothiocyanate

[0090] Para-aminobenzoic acid (0.2 moles; 26 grams of 99 percent purefrom Aldrich Chemical, Milwaukee, Wis.) was dissolved in acetone (400mL) at room temperature (about 20 degrees Celsius). Activated carbon(about 5 grams; Darco® G60) was added, and the mixture was stirred(magnetic stir bar) for 5 to 10 minutes. The entire solution wasfiltered, yielding a much lighter-colored solution of p-aminobenzoicacid than was initially formed.

[0091] Sodium acetate (0.3 moles, 25 grams; dissolved in about 200 mL ofdeionized water) was added to the filtrate, now contained in a 4 litervacuum flask. A vacuum was applied to the flask with an intermediate dryice/acetone trap between the 4 liter flask and the vacuum pump. Reducedpressure was maintained until much of the original acetone hadevaporated off of the p-aminobenzoic acid solution (down to about 300 mLvolume). The flask was chilled to between about zero and five degreesCelsius.

[0092] Thiophosgene (about 40 grams of neat red liquid) were added inone portion to the cooled, acetone-stripped slurry of p-aminobenzoicacid, while stirring rapidly with an overhead paddle stirrer. A tanprecipitate formed almost immediately upon addition of the thiophosgene,along with considerable foaming. After the foaming subsided (about 10minutes), the insoluble precipitate was filtered and dried in vacuountil it was a free-flowing powder.

[0093] The crude product was re-crystallized from hot (about 80° C.)glacial acetic acid to yield light yellow needles of 4-carboxyphenylisothiocyanate (about 16 grams) after drying in vacuo. Considerableproduct remained in the mother liquor, which was not recovered.

[0094] Elemental analysis of the yellow crystals yielded: Carbon (found53.38 percent, theory 53.63 percent); hydrogen (found 2.76 percent,theory 2.79 percent); nitrogen (found 7.58 percent, theory 7.82percent). The crystals darkened but did not melt at 220° C. The infraredspectrum of the crystals was consistent with that expected for4-carboxyphenyl isothiocyanate.

EXAMPLE 2 Synthesis of 4-Isothiocyanobenzoyl Azide

[0095] A portion of the 4-carboxyphenyl isothiocyanate (15 grams) fromExample 1 was suspended in dry methylene chloride (200 mL) in a 1 literside-arm vacuum flask, along with pyridine (16 grams; 0.2 M), phenyldichlorophosphate (Aldrich Cat. No. P.2, 238-9; 0.1 M) and sodium azide(6.5 grams; 0.1 M). The mixture was stirred overnight (about 15 hours)at room temperature. The stirred mixture was then washed in a separatoryfunnel with water (200 mL) and then sulfuric acid (200 mL of 0.1 NH₂SO₄). The acid-washed methylene chloride layer was dried withanhydrous magnesium sulfate (MgSO₄).

[0096] The dried methylene chloride reaction solution was evaporatedunder vacuum in a rotary evaporator at room temperature or lower (lessthan or equal to about 20° C.). The resulting light tan crystals weredissolved in a minimum of ethyl ether at room temperature. There-crystallization solution was treated with activated carbon (Darco®G60) and filtered. The resulting light-colored solution was evaporatedto dryness in vacuo at a temperature not exceeding 20° C. Nearly whitecrystals were obtained melting at 68-72° with evolution of nitrogen,consistent with azide decomposition.

[0097] The elemental analysis of the 4-isothiocyanobenzoyl azidecrystals gave the following results: Carbon (found 46.85 percent, theory47.05); Hydrogen (found 2.09 percent; theory 1.96 percent); Nitrogen(found 26.87 percent; theory 27.45 percent). The infrared spectrum ofthe crystals (FTIR) conformed to 4-isothiocyanobenzoyl azide (e.g.strong, broad band from about 2000 to about 2200 cm⁻¹ is from N═C═O andN═C═S stretching modes; and a strong absorption due to azide at about980-1000 cm⁻¹) The compound is stable at room temperature but was storedin the freezer.

EXAMPLE 3 Synthesis of 4-Isothiocyanophenyl Isocyanate and PEG Reagent

[0098] The nearly white 4-isothiocyanobenzoyl azide from Example 2 wasthermally decomposed at about 75°-104° C. (Curtius Rearrangement)smoothly and quantitatively as a solution in dry refluxing toluene. Theresulting 4-isothiocyanophenyl isocyanate (also known as4-isocyanophenyl isothiocyanate, shown below) product is represented bythe following chemical formula. The 4-isocyanophenyl isothiocyanate wasnot isolated, but reacted as formed in situ with the hydroxyl-containingPEG according to the following description. An infrared spectrum of thecomposition containing 4-isocyanophenyl isothiocyanate showed thedecrease in the azide band at about 980-1000 cm⁻¹ along with increasedcomplexity of the absorbance in the N═C═O and N═C═S region of thespectrum from about 2000 to about 2300 cm⁻¹.

[0099] A 2-liter, 3-necked flask was equipped with a thermometer,overhead stirrer with motorized drive, heating mantle with rheostat,short column with Dean-Stark trap and condenser. The flask containedtoluene solvent (500 mL) charged with methoxypolyethylene glycol havinga low diol content (“PEG methyl ester”, MW 5000; 50 grams; 10 mL;manufactured by NOF Corp. Japan, available as catalog No. 2M2000H01 fromShearwater Corp., Huntsville, Alabama; HO—CH₂CH₂—(OCH₂CH₂)_(x)—O—CH₃,where x is about 112).

[0100] The PEG methyl ether (mPEG) mixture was stirred and heated toreflux (about 104° C.) and any water present was azeotropically removedas it accumulated in the Dean-Stark trap. When no additional waterformed in the Dean-Stark trap, heating was discontinued and the reactionvessel and its contents were cooled under a dry N₂ blanket until theinternal reaction solution temperature was less than about 60° C.

[0101] After the reaction solution temperature fell to less than about60° C., p-isothiocyanobenzoyl azide (0.5 grams from Example 2) was addedto the reaction as a solid (through thermometer port), and heating wasresumed. A brisk and steady stream of nitrogen exited the reactor duringthe initial about 15 to about 30 minutes after heating was resumed.Heating was continued (reflux) for an additional hour after nitrogenevolution ceased, as monitored by bubble trap at the exit of condenser.Heating was then discontinued and the flask and contents allowed to coolto room temperature overnight (about 16 hours), providingmPEG-O-p-carbamoylphenyl isothiocyanate.

[0102] Workup and purification of phenylisothiocyanate-activated mPEG ofthis example was accomplished by concentrating the reaction solutionunder vacuum by means of a rotary evaporator. The remaining viscous oilwas triturated with anhydrous ethyl ether to induce crystallization ofthe activated mPEG (nearly white). The ether trituration inducescrystallization and extracts excess, unreacted p-isothiocyanophenylisocyanate.

[0103] The crude product was filtered and dried in vacuo (yield 51grams). The dried crude product was dissolved and stirred with water(500 mL deionized). A slight amount of water-insoluble matter wasfiltered out of the phenylisothiocyanate-activated mPEG using 0.2 μgglass mat filter paper, yielding a clarified filtrate. The clarifiedfiltrate was extracted with methylene chloride (2×200 mL) in aseparatory funnel. The methylene chloride extract was dried withanhydrous magnesium sulfate, and filtered. The dried, filtered extractwas concentrated under vacuum (rotary evaporator) to a viscous oil.Product was precipitated by addition of diethyl ether. Therecrystallized phenylisothiocyanate-activated m PEG product was filteredand dried under vacuum to yield a white solid (48 grams). Elementalanalysis of the phenylisothiocyanate-activated m PEG: Carbon (found54.09 percent, theory 54.5 percent); Hydrogen (found 8.95 percent;theory 9.09 percent); Nitrogen (found 0.32 percent, theory 0.5 percent).

EXAMPLE 4 Synthesis of Bifunctional PEG Reagent

[0104] 4-isothiocyanobenzoyl azide is decomposed and reacted in situwith the two alcohol moieties of a PEG diol compound to form a PEGisothiocyanate crosslinking reagent. A 2-liter, 3-neck flask is equippedwith a thermometer, overhead stirrer with motorized drive, heatingmantle with rheostat, short column with Dean-Stark trap and condenser.The flask contains toluene solvent (500 mL) charged with polyethyleneglycol (“PEG diol”, MW 5000; 25 grams, HO—CH₂CH₂—(OCH₂CH₂)_(x)—O—H,where x is about 112).

[0105] The PEG diol/toluene mixture is stirred and heated to reflux(˜104° C.) and any water present is azeotropically removed as itaccumulates in the Dean-Stark trap. When no additional water forms inthe Dean-Stark trap, heating is discontinued and the reaction vessel andits contents are cooled under a dry N₂ blanket until the internalreaction solution temperature is less than about 60° C.

[0106] After the reaction solution temperature falls to less than about60° C., p-isothiocyanobenzoyl azide (0.5 grams from Example 2) is addedto the reaction, and heating is resumed. Nitrogen evolution ismonitored. Heating is continued at reflux for an additional hour afterthe nitrogen evolution ceases. Heating is then discontinued and theflask and contents cooled, providing the PEG isothiocyanate crosslinkingreagent shown below.

[0107] The PEG crosslinking reagent product is dried down, trituratedwith ether and crystallized. The PEG crosslinking reagent is dissolvedin water, extracted into methylene chloride, dried and re-crystallized.

EXAMPLE 5 Preparation of 3,5-Dicarboxyphenyl Isothiocyanate

[0108] Acetone (1.4 L), sodium acetate in water (1.0 L of 1 M) and5-aminoisophthalic acid (50 g; Aldrich Catalog No. 18,627-9) were addedto a flask (4 L) equipped with a stirrer. The pH of the resulting slurrywas adjusted to 6.8-7.0 by dropwise addition of a 50 percent sodiumhydroxide aqueous solution. An additional 500 mL of water yielded ahomogeneous, yellow solution of 5-aminoisophthalic acid which wastreated with activated carbon, filtered and chilled to 5-10° C. by theaddition of ice cubes.

[0109] To the rapidly stirred and chilled solution was then added 25.0mL of thiophosgene liquid (Aldrich Catalog No. 1,515-0) in one shot.After stirring for 30 minutes, concentrated hydrochloric acid was addeddropwise until the pH of the resulting reaction was 3-4, causing thecrude product to precipitate.

[0110] Two recrystallizations from a minimum of 85° C. acetic acidyielded pale yellow crystals of 3,5-dicarboxyphenyl isothiocyanate.Elemental and IR analyses conform to the expected results. Elementalanalysis of the 3,5-dicarboxyphenylisothiocyanate: Carbon (found 46.34percent, theory 48.43 percent); Hydrogen (found 3.16 percent; theory2.24 percent); Nitrogen (found 4.89 percent, theory 6.27 percent).

EXAMPLE 6 Preparation of 3,5-Di-Acylazidophenyl Isothiocyanate

[0111] The di-carboxylic acid compound from the preceding Example,3,5-dicarboxyphenyl isothiocyanate (8.9 g, 0.04 moles), was reacted withpyridine (16 g; 0.2 moles) and phenyl dichlorophosphate (226 M; 0.1moles) and sodium azide (6.56 g; 0.1 moles) in methylene chloride (300mL) in the manner described in Example 2, above. Recrystallization frommethylene chloride produced cream-colored, crystalline powder of thedi-acylazidophenyl isothiocyanate (6 g; melting point 92-94° C. withevolution of nitrogen). The infrared and elemental analyses conformed tothe expected results. A strong azido absorbance at about 1192 cm⁻¹ wasobserved. Elemental analysis of the 3,5-di-acylazidophenylisothiocyanate: Carbon (found 39.99 percent, theory 39.56 percent);Hydrogen (found 1.42 percent; theory 1.09 percent); Nitrogen (found32.21 percent, theory 35.89 percent).

EXAMPLE 7 Preparation of 3,5-Di-Carbamoyl Methoxy Polyethylene Glycol

[0112] The 3,5-di-acylazido phenylisothiocyanate from Example 6 (1.26 g;about 0.005 moles) was thermally decomposed at about 92°-94° C. (CurtiusRearrangement) in dry refluxing toluene in a three-necked flask (2 L)equipped with a mechanical stirrer, a Dean-Stark trap and a condenser.The resulting 3,5-di-isocyanophenyl isothiocyanate product was notisolated, but reacted as formed in situ with methoxy polyethylene glycol(50 g; 0.01 moles) that had been prepared as described in Example 2,above. The reaction solution was heated with evolution of nitrogen andthen refluxed for an additional hour after nitrogen evolution ceased.

[0113] After cooling, solvent was removed and the product crystallizedfrom the viscous oil by trituration with anhydrous ether. The solidproduct was further purified by extracting the solid from an aqueoussolution into methylene chloride (dried with anhydrous magnesiumsulfate) re-concentrated and triturated with anhydrous ether. Thepurified 3,5-(di-carbamoyl mPEG) phenyl isothiocyanate (48.3 g) wasnearly white.

EXAMPLE 8 Synthesis of a Cellulose Reagent

[0114] An insoluble reagent for linking amine groups of targetmolecules, such as proteins, is prepared from cellulose. The activatedcellulose linking reagent is stored as a dry reagent with a relativelylong shelf life.

[0115] Cellulose has a plurality of primary hydroxy groups. Cellulose issoaked overnight at 20-60° C. in a solution of p-isothiocyanophenylisocyanate prepared in situ as described above in Example 3. Theactivated cellulose surface is washed repeatedly with fresh toluene anddried.

EXAMPLE 9 Pegylation of Hemoglobin

[0116] A para-(methoxy polyethylene glycol 5000 carbamic acid)derivative of phenyl isothiocyanate (mPEG reagent) was prepared usingthe methods described in Examples 1-3. Hemoglobin was purified fromhuman red blood cells through methods known in the art. The accessiblehemoglobin α-amino group was reacted with the m-PEG reagent permittingan mPEG reagent solution (1 mM) in pH 7.4 phosphate buffered salinecontaining hemoglobin (0.5 mM) to remain overnight (about sixteen hours)in a cold room (about 4° C.).

[0117] The reaction product was dialyzed against 10 mM potassiumphosphate, pH 6.5, and subjected to purification on a CM-cellulosecolumn. Analysis of the resulting pegylated hemoglobin by methodsdescribed in Belur N. Manjula, et al., J. Biol. Chem., 275(8):5527-5534(2000) reveal a molecular radius of 5.2 consistent with a hemoglobinmodified with four molecules of methoxy PEG 5000 per hemoglobintetramer.

[0118] The pegylated hemoglobin obtained had a slightly higher oxygenaffinity as compared to unmodified hemoglobin. Thus the molecular radiusmeasurements are consistent with the modification of the four alphaamino groups of the hemoglobin tetramer.

[0119] The disclosures of each of the patents and articles cited hereinis incorporated by reference. The use of the article “a” or “an” in aclaim hereinbelow is intended to include one or more, unless otherwisespecifically stated.

[0120] From the foregoing, it will be observed that numerousmodifications and variations can be effected without departing from thetrue spirit and scope of the present invention. It is to be understoodthat no limitation with respect to the specific examples presented isintended or should be inferred. The disclosure is intended to cover bythe appended claims modifications as fall within the scope of theclaims.

What is claimed is:
 1. A compound represented by the formula

where n=1 or 2; R is —NHC(O)—O—M, —NCO or —C(O)N₃; M is a reactedalcohol-containing macromolecule; and R is in a para-, meta- or di-metaposition relative to —NCS.
 2. The compound according to claim 1, whereinM is a reacted polyethylene glycol or polysaccharide.
 3. The compoundaccording to claim 2 wherein the polysaccharide is dextran, cellulose,starch or agarose.
 4. The compound according to claim 1 where R is —NCO.5. The compound according to claim 1 where R is —C(O)N₃.
 6. The compoundaccording to claim 1 where R is —NHC(O)—O—M.
 7. The compound accordingto claim 6 represented by the formula

wherein M is the reacted methoxy polyethylene glycol—CH₂CH₂—(OCH₂CH₂)_(x)—O—CH₃; and x is an average value that is about 5to about
 500. 8. The compound according to claim 6,

wherein said compound is represented by the formula above and x is anaverage value that is about 5 to about
 500. 9. A compound represented bythe formula

where B is a reacted amino group-containing biomolecule; R is—NHC(O)—O—M; where n=1 or 2; M is a reacted alcohol-containingmacromolecule; and —R is para, meta or di-meta relative to —NHC(S)—NH—B.10. The compound according to claim 9 where M is methoxy polyethyleneglycol.
 11. The compound according to claim 9 wherein said macromoleculeM is a hydroxy-containing surface.
 12. The compound according to claim 9wherein said biomolecule B is streptavidin.
 13. The compound representedby the formula

where M is a reacted alcohol-containing macromolecule.
 14. The compoundaccording to claim 13 where M is polyethylene glycol.
 15. The compoundaccording to claim 14 represented by the chemical formula

wherein x is an average value that is about 5 to about
 500. 16. Acompound represented by the chemical formula

where B and B′ are the same or different reacted amino group-containingbiomolecules, and M is a reacted alcohol-containing macromolecule. 17.The compound according to claim 16 where M is polyethylene glycol.
 18. Amethod for making a macromolecule M that is linked to a biomolecule Bcomprising the following steps: (a) providing a linking reagentrepresented by the formula

where n=1 or 2; R is —NHC(O)—O—M; M is a reacted alcohol-containingmacromolecule; and R is in a para-, meta- or di-meta position relativeto —NCS; (b) providing an amine-containing biomolecule B in an admixturewith the linking reagent provided in step (a) to form a linking mixture;and (c) maintaining said linking mixture for a time period sufficient toform a urethane compound represented by the chemical formula

where n=1 or 2; R is —NHC(O)—O—M; M is a reacted alcohol-containingmacromolecule; and R is in a para-, meta- or di-meta position relativeto —N(H)C(S)N(H)—B thereby making a macromolecule M that is linked to abiomolecule B.
 19. The method according to claim 18 wherein saidmacromolecule M is a polyethylene glycol.
 20. The method according toclaim 18 wherein said biomolecule B is a polypeptide.